Method and composition for the treatment of cancer

ABSTRACT

We have surprisingly discovered that the administration of a G1 and/or S phase drug such as β-lapachone in combination with a G2/M drug such as a taxame derivative such as paclitaxel resulted in an unexpected greater than additive (i.e., synergistic) reduction in the number of tumors (and tumor volume) as compared with the administration of these agents alone. In addition, no signs of toxicity or weight loss were observed. The present invention relates to a method for treating a mammalian tumor using combinations such as a taxane derivative, preferably paclitaxel, and a β-lapachone, or a derivative or analog thereof.

This application is a 371 of PCT/US00/10169, filed Apr. 14, 2000, andclaims the benefit of Provisional Application No. 60/129,261, filed Apr.14, 1999.

BACKGROUND OF THE INVENTION

The treatment of cancer has thus far proved problematic. While “cancers”share many characteristics in common, each particular cancer has its ownspecific characteristics. Genetics and environmental factors have acomplex interplay in severity and prognosis of treatment. Thus,treatment must be carefully tailored.

Certain pharmaceutical treatments have proved useful for one form ofcancer, but not others (Hollad and Frei, et al, Cancer Medicine, 4th ed.Publisher Williams & Wilkens). Other treatments such as radiation, whilepartially useful for a range of cancers, do not typically result in acomplete cure. Indeed, given the severity of many cancers and themortality rate, a drug can be deemed successful if it improves qualityof life, e.g., by delaying growth of tumors, or prolongs life—withoutactually curing the condition. Thus, in many circumstances, anindividual is treated with a compound or combination of treatments thatcan eliminate 90-95% of the malignant cells, but the remaining cells canregrow and metastasize, ultimately resulting in death. Among cancerswith particularly poor ultimate prognoses is ovarian cancer.

Combination therapies, while desirable, are a hit or miss proposition.The treatments are typically not addictive. In many cases, cross effectsand treatment load can result in lower effectiveness for thecombinations, than either treatment alone. Problems encountered includemultiple drug resistance (MDR), where the malignant cell in essencepumps the cytotoxic compounds and other compounds out of the cell,thereby preventing continued useful treatment of the cancer.

There are a number of cytotoxic agents that are currently being used orstudied for the treatment of cancer. One of these, Paclitaxel, (alsoreferred to as TAXOL®) was first identified in 1971 by Wani andcollaborators (Wani MC et al., 1971 J. Am. Chem. Soc., 93: 2325-2327)following a screening program of plant extracts of the National CancerInstitute. This complex diterpene shows cytotoxic activity againstseveral types of tumors and is presently used in the treatment of somecancers such as ovarian and breast cancers. Clinical studies suggestthat TAXOL® could eventually be used in the treatment of over 70% ofhuman cancers.

Paclitaxel differs from other cytotoxic drugs by its unique mechanism ofaction. It interferes with cell division by manipulating the molecularregulation of the cell cycle. Paclitaxel binds to tubulin, the majorstructural component of microtubules that are present in all eukaryoticcells. Unlike other antimitotic agents such as vinca alkaloids andcolcichine, which inhibit the polymerization of tubulin, paclitaxelpromotes this assembly of tubulin and stabilizes the resultingmicrotubules. This event leads to the interruption of cell division, andultimately to cell death.

The antitumor property of taxoid compounds has also lead to thegeneration of new anticancer drugs derived from taxanes. Taxotere™ (soldby Rhône-Poulenc Rorer), which is produced from 10-deacetylbaccatin IIIby hemisynthesis, is currently used in the treatment of ovarian andbreast cancers.

While agents such as TAXOL® and Taxotere have made an advance in thetreatment of metastatic ovarian and metastatic breast cancer, themajority of those treated still ultimately succumb to these diseases.β-lapachone, a quinone, is derived from lapachol (a naphthoquinone)which can be isolated from the lapacho tree (Tabebuia avellanedae), amember of the catalpa family (Bignoniaceae). Like camptothecin andtopotecan, β-lapachone inhibits DNA Topoisomerase I (Li, C. J., et al.,J. Biol. Chem., 1993). This compound has been found to be effectiveagainst several types of cancer cells in vitro, including lung, breast,colon and prostate cancers and malignant melanoma (Li, C. J., et al.,Cancer Research 55:3712-3715 (1995) and unpublished data).

β-lapachone works by disrupting DNA replication. Topoisomerase I is anenzyme that unwinds the DNA that makes up the chromosomes. Thechromosomes must be unwound in order for the cell to use the geneticinformation to synthesize proteins; β-lapachone keeps the chromosomeswound tight, and so the cell can't make proteins. As a result, the cellstops growing. Because cancer cells are constantly replicating andcircumvent many mechanisms that restrict replication, as is the casewith normal cells, they are more vulnerable to topoisomerase inhibitionthan are normal cells. However, treatment with these compounds is alsoonly partially successful—inhibiting and delaying growth of themalignant cells.

No single drug or drug combination is curative for advanced metastatic.cancer and patients typically succumb to the cancers in several years.Thus, new drugs or combinations that can prolong onset oflife-threatening tumors and/or improve quality of life by furtherreducing tumor-load are very. important.

SUMMARY OF THE INVENTION

We have surprisingly discovered that the administration of a compoundthat targets cells at G1 and/or S phase such as a topoisomerase Iinhibitor such as β-lapachone in combination with a compound thattargets such cells at G2/M phase, e.g., a taxane derivative such aspaclitaxel resulted in an unexpectedly greater than additive (i.e.,synergistic) reduction in the number of tumors (and tumor volume in amammal with metastatic tumors) as compared with the administration ofthese agents alone. Furthermore, the tumors did not grow back in severalmonths of observation. In addition, no signs of toxicity or weight losswere observed in mammals so treated.

Accordingly, the present invention relates to a method for treating amammalian tumor using a combination of a G2/M phase drug including, butnot limited to, taxane, its derivatives and analogs, more preferablypaclitaxel, and a G1 and/or S phase drug, preferably P-lapachone, or aderivative or analog thereof.

A list of two representative compounds is described in Table 1, infra.The combination of the present invention is particularly advantageous inthe treatment of patients who have chemotherapeutically refractivemetastatic cancer. The method of the present invention comprisesadministering to the mammal in combination an effective amount of a G1and S phase drug, a G1 phase drug, a S phase drug, in combination with aG2/M drug. Preferably, the combination is (1) a topoisomerase Iinhibitor such as β-lapachone or its derivatives or analog thereof; and(2) taxane, its derivatives or analogs and pharmaceutically acceptablesalts thereof.

As used herein, the phrase “taxane derivative” means any taxane which isor may be used in cancer chemotherapy due to its antineoplasticproperties. TAXOL® is a preferred taxane derivative.

As further used herein, the phrase “β-lapachone” means lapachone(3,4-dihydro-s,3-dimethyl-2H-naphthol[1,3-b] pyran-5,6-clone) andderivatives and analogs thereof. Preferred derivatives and analogs arediscussed below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate the results of Experiment 1.

FIGS. 2A and 2B illustrate the results of Experiment 2.

FIG. 3 is a photograph of the control group showing hundreds of tumornodules some of which have fused together to form large tumor masses(e.g., arrow 2).

FIG. 4 is a photograph of the β-lapachone treated group showing severaltumor nodules (e.g., arrow 2).

FIG. 5 is a photograph of the paclitaxel treated group showing severaltumor nodules (e.g., arrow 2).

FIG. 6 is a photograph of the combination treated group. No tumornodules are visible. It is also important to note the color of theperitoneal lining. Unlike the lining seen in FIGS. 3-5, the color of thelining in the combination group is not bright red but more like thatseen in healthy mice. This provides an indication that tumor growth andtumor induced angiogenesis was not occurring.

FIGS. 7A and 7B are photographs showing combination treatment on aprostate tumor. FIG. 7A is the control. FIG. 7B is the combination.

DETAILED DESCRIPTION

This invention provides for advantageous combination therapies forcancers, including, but not limited to, breast, ovarian and prostatecancer using methods which employ administration of a G1 and/or S phasecompound with a G2/M phase compound.

In one embodiment, the invention is directed to a method for treating asubject having malignant cells or inhibiting further growth of suchmalignant cells by using a compound that targets such cells at G1 and/orS phase checkpoints, simultaneously with/or followed by using a drugthat acts at G2/M checkpoints. Individual compounds satisfying thiscriteria are known to those of ordinary skill in the art. For example,β-lapachone and its derivatives are G1 and S phase drugs. Whereas taxoland its derivatives are G2/M drugs. A list of representative compoundsis set forth below in Table 1:

TABLE 1 Type Category Compound Name Chemical Formula 1. G1 and S phasedrug β-lapachone Reduced β-lapachone 2. G1 phase drugs Lovastatin[1S[1α(R*),3α7β,8β S*,4S*),8αβ]]- Methylbutanoic acid1,2,3,7,8,8a-hexahydro-3,7- dimethyl-8-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2- yl)ethyl]-1-naphthalenyl ester Mimosineα-Amino-3-hydroxy-4-oxo- 1(4H)-pyridine propanoic acid Tamoxifen[Z]-2-[4-(1,2-Diphenyl-1- butenyl)-phenoxy]N,N- dimethylethanamine 3. Sphase drugs Gemcitabine 2′,2′difluorodeoxycytidine 5-FU 5-fluorouracilMTX Methotrexate; N-[4-[[(2,4- Diamino-6- pteridinyl)methyl]]methylamino]benzoyl]-L-glutamic acid 4. G2/M drugs (i) Microtubule-targeting Taxol5-beta,20-epoxy-1,2-alpha,4,7- beta,10-beta,13-alpha-hexahydroxy-tax-11-en-9-one 4,10-diacetate 2-benzoate 13- ester with(2R,3S)-N-benzoyl- 3-phenyl-isoserine Docetaxel N-debenzoyl-N-tert-butoxycarbonyl-10-deacetyl taxol Epothilone Epithilone Polyketides A, B,C or D (desoxy-epothilne) Vincristin 22-Oxovicaleukoblastine VinblastinVincaleukoblastine Nocodazole 2-Benzimidazolecarbamic acid,5-(2-thenoyl)-, methyl ester Navelbine Vinorelbine (ii) TopoisomeraseTeniposide VM-26; [5R-5α,5αβ,8aα,9β (R*)]]-   Poisons5,8,8a,9-tetrahydro-5-(4- hydroxy-3,5- dimethoxyphenyl)-9-[[4,6-O-(2-thienylmethylene)-β-D- glucopyranosyl]oxy]furo[3′,4′:6,7]naphtho[2,3-d]-1,3-dioxol- 6(5aH)-one Etoposide VP-16; 4′-Demethylepipodophyllotoxin ethylidene-B-D-glucoside AdriamycinDoxorubicin; 14-Hydroxydaunomycin Camptothecin Daunorubicin Cerubidin;Leukaemomycin C; Rubidomycin; Rubomycin C Dactinomycin Actactiomycin AIV; Actinomycin C1; Actinomycin- [threo-val-pro-sar-meval] MitoxantroneAmsacrine Epirubicin Idarubicin Idamycin; 4-demethoxy- daunorubicin

The combinations of the present invention are particularly advantageousas shown by the example with β-lapachone and taxol where synergisticresults were obtained. Molecular changes underlying cell cycle delay atmultiple checkpoints, for example G1 and/or S phase and G2/M phase, canfor example result in the synergistic induction of apoptosis inmalignant cells. Although not wishing to be bound by theory, it isbelieved that the synergistic effect is mediated by inhibition of cdc2kinases and upregulation of p21. p21 controls G1 and S phase checkpoints(Elledge, S. J. (1996) Science 274, 1664-1672), and is involved in theregulation of the G2/M checkpoint (Hartwell L. H. et al., M. B. (1994)Science, 266, 1821-1828). Cell cycle checkpoints are also regulated bycdc2 kinases and their inhibitors (Elledge, S.J. (1996) Science 274,1664-1672 and Nurse, P. (1997) Cell 91, 865-867).

Preferably, the G1 and/or S phase compounds are administered prior to,or simultaneously with compounds that target a cell at the G2/M phasecheckpoint. More preferably, the compounds are administered prior to thecompounds that target a cell at the G2/M checkpoint.

Preferred G1 and/or S phase checkpoint targeting compounds include G1and/or S phase drugs (for example, β-lapachone), G1 phase drugs (forexample, lovastatin, mimosine, tamoxifen, etc.) and S phase drugs (forexample, gemcitabine, 5-FU, MTX, etc.). β-lapachone, its derivatives andanalogs are more preferred.

Further, G1 and/or S phase checkpoint targeting drugs includederivatives of reduced β-lapachone. Reduction of β-lapachone has beenshown to be an essential component of β-lapachone activity (see J. J.Pink et al (2000) J Biol. Chem. 275: 5416-5424). Preferably, G1 and/or Sphase checkpoint targeting drugs would further include reducedβ-lapachone, i.e reduced β-lapachone derivatives or analogs and/or acombination of β-lapachone derivatives or analogs (quinine form) withreduced β-lapachone derivatives or analogs (hydroquinone form). Mostpreferably, the reduced β-lapachone, its derivatives or analogs wouldinclude modified hydroquinone groups in the reduced β-lapachone as shownin Formula Ia where the R′ and/or R″ group(s) are converted to, forexample, succinates, amino acids etc.

Preferred G2/M phase checkpoint targeting compounds includemicrotuble-targeting drugs (for example, taxol, docetaxel, vincristin,vinblastin, nocodazole, epothilones, navelbine, etc.) and topoisomerasepoisons (for example, teniposide, etoposide, adriamycin, camptothecin,daunorubicin, dactinomycin, mitoxantrine, amsacrine, epirubicin,idarubicin, etc.).

Epothilones (epothilone polyketides) are microtubule targeting drugswhich stabilize microtubules by means of the same mechanisms as taxol(see Litang, et al. (2000) Science 287, 640-642). The epothilones areadvantageous as they are effective against taxol-resistant tumors andare sufficiently water soluble. Epothilones A and B are the mostabundant in nature and 12,13-desoxy-epothilone B (epothilone D) has thehighest therapeutic index. Epothilones (A, B, C, D or mixtures thereof)can be used in combination with β-lapachone and this could in asynergistic induction of apoptosis in malignant cells which is similarto the combination of β-lapachone and taxol, as described earlier. Forthe purpose of this invention, epothilone would refer to epothilones A,B, C or D (desoxy-epothilone).

Preferred combinations include:

β-lapachone with taxol; β-lapachone with docetaxel; β-lapachone withvincristin; β-lapachone with vinblastin; β-lapachone with nocodazole;β-lapachone with teniposide; β-lapachone with etoposide; β-lapachonewith adriamycin; β-lapachone with epothilone; β-lapachone withnavelbine; β-lapachone with camptothecin; β-lapachone with daunorubicin;β-lapachone with dactinomycin; β-lapachone with mitoxantrone;β-lapachone with amsacrine; β-lapachone with epirubicin; or β-lapachonewith idarubicin.

Reduced β-lapachone with taxol; reduced β-lapachone with docetaxel;reduced β-lapachone with vincristin; reduced β-lapachone withvinblastin; reduced β-lapachone with nocodazole; reduced β-lapachonewith teniposide; reduced β-lapachone with etoposide; reduced β-lapachonewith adriamycin; reduced β-lapachone with epothilone; reducedβ-lapachone with navelbine; reduced β-lapachone with camptothecin;reduced β-lapachone with daunorubicin; reduced β-lapachone withdactinomycin; reduced β-lapachone with mitoxantrone; reduced β-lapachonewith amsacrine; reduced β-lapachone with epirubicin; or reducedβ-lapachone with idarubicin.

Lovastatin with taxol; lovastatin with docetaxel; lovastatin withvincristin; lovastatin with vinblastin; lovastatin with nocodazole;lovastatin with teniposide; lovastatin with etoposide; lovastatin withadriamycin; lovastatin with epothilone; lovastatin with navelbine;lovastatin with camptothecin; lovastatin with daunorubicin; lovastatinwith dactinomycin; lovastatin with mitoxantrone; lovastatin withamsacrine; lovastatin with epirubicin; or lovastatin with idarubicin.

Mimosine with taxol; mimosine with docetaxel; mimosine with vincristin;mimosine with vinblastin; mimosine with nocodazole; mimosine withteniposide; mimosine with etoposide; mimosine with adriamycin; mimosinewith epothilone; mimosine with navelbine; mimosine with camptothecin;mimosine with daunorubicin; mimosine with dactinomycin; mimosine withmitoxantrone; mimosine with amsacrine; mimosine with epirubicin; ormimosine with idarubicin.

Tamoxifen with taxol; tamoxifen with docetaxel; tamoxifen withvincristin; tamoxifen with vinblastin; tamoxifen with nocodazole;tamoxifen with teniposide; tamoxifen with etoposide; tamoxifen withadriamycin; tamoxifen with epothilone; tamoxifen with navelbine;tamoxifen with camptothecin; tamoxifen with daunorubicin; tamoxifen withdactinomycin; tamoxifen with mitoxantrone; tamoxifen with amsacrine;tamoxifen with epirubicin; or tamoxifen with idarubicin.

Gemcitabine with taxol; gemcitabine with docetaxel; gemcitabine withvincristin; gemcitabine with vinblastin; gemcitabine with nocodazole;gemcitabine with teniposide; gemcitabine with etoposide; gemcitabinewith adriamycin; gemeitabine with epothilone; gemcitabine withnavelbine; gemcitabine with camptothecin; gemcitabine with daunorubicin;gemcitabine with dactinomycin; gemcitabine with mitoxantrone;gemcitabine with amsacrine; gemcitabine with epirubicin; or gemcitabinewith idarubicin.

5-FU with taxol; 5-FU with docetaxel; 5-FU with vincristin; 5-FU withvinblastin; 5-FU with nocodazole; 5-FU with teniposide; 5-FU withetoposide; 5-FU with adriamycin; 5-FU with epothilone; 5-FU withnavelbine; 5-FU with camptothecin; 5-FU with daunorubicin; 5-FU withdactinomycin; 5-FU with mitoxantrone; 5-FU with amsacrine; 5-FU withepirubicin; or 5-FU with idarubicin.

MTX with taxol; MTX with docetaxel; MTX with vincristin; MTX withvinblastin; MTX with nocodazole; MTX with teniposide; MTX withetoposide; MTX with adriamycin; MTX with epothilone; MTX with navelbine;MTX with camptothecin; MTX with daunorubicin; MTX with dactinomycin; MTXwith mitoxantrone; MTX with amsacrine; MTX with epirubicin; or MTX withidarubicin.

The combination of the present invention results in a surprising synergywhich is beneficial in reducing tumor burden load and/or regressingtumor growth, especially in patients with metastatic disease.

Preferably, the cancers treated are breast, ovarian, prostate, lung,colon and melanoma. More preferably, the cancer is ovarian.

The compounds can be administered by any means known in the art. Suchmodes include oral, rectal, nasal, topical (including buccal andsublingual) or parenteral (including subcutaneous, intramuscular,intravenous and intradermal) administration.

For ease to the patient oral administration is preferred. However,typically oral administration requires a higher dose than an intravenousadministration. Thus, depending upon the situation—the skilled artisanmust determine which form of administration is best in a particularcase—balancing dose needed versus the number of times per monthadministration is necessary.

In administering the compounds one can use the normal dose of eachcompound individually. However, preferably one uses a lowerlevel—typically 75% or less of the individual amount, more preferably50% or less, still more preferably 40% or less.

The individual components will be addressed in more detail below.

One preferred component of the combination therapy described is a taxanederivative. The taxanes are a family of terpenes, including, but notlimited to paclitaxel and docetaxel (Taxatere), which were derivedprimarily from the Pacific yew tree. Taxus brevifoilia, and which haveactivity against certain tumors, particularly breast and ovarian tumors.Paclitaxel is a preferred taxane. It is considered an antimicrotubuleagent that promotes the assembly of microtubules from tubulin dimers andstabilizes microtubules by preventing depolymerization. This stabilityresults in the inhibition of the normal dynamic reorganization of themicrotubule network that is essential for vital interphase and mitoticcellular functions. The term “paclitaxel” includes both naturallyderived and related forms and chemically synthesized compounds orderivatives thereof with antineoplastic properties includingdeoxygenated paclitaxel compounds such as those described in U.S. Pat.No. 5,440,056, herein incorporated by reference, and that sold is soldas TAXOL® by Bristol-Myers Oncology. Chemical formulas for paclitaxelare known and disclosed in U.S. Pat. No. 5,440,056. In addition toTAXOL®, other derivatives are well known, e.g., those mentioned in“Synthesis and Anticancer Activity of TAXOL® other Derivatives,” D.G.I.Kingston et al., Studies in Organic Chemistry, vol. 26, entitled “NewTrends in Natural Products Chemistry” (1986), Atta-ur-Rahman, P. W. leQueene, Eds. (Elvesier, Amsterdam 1986), pp. 219-235. Still other taxanederivatives are known in the art and include those, for example,disclosed in U.S. Pat. Nos. 5,773,461; 5,760,072; 5,807,888; and5,854,278.

The G2/M compound such as the taxane derivative may be administered inany manner found appropriate by a clinician in generally acceptedefficacious dose ranges such as those described in the Physician DeskReference, 53th Ed. (1999), Publisher Edward R. Barnhart, New Jersey(“PDR”) for paclitaxel.

In general, the G2/M compound such as the taxane derivative isadministered intravenously at dosages from about 135 to about 300 mg/m²,preferably from about 135 to about 175 mg/m², and most preferably about175 mg/m². It is preferred that dosages be administered over a timeperiod of about 1 to about 24 hours, typically over a period of about 3hours. Dosages can be repeated from 1 to about 4 weeks or more,preferably from about 2 to about 3 weeks.

The drug may be administered in any form such as by injection or oralforms. Liposome formulations, for example, have been described See, e.g.U.S. Pat. No. 5,424,073, which is herein incorporated by reference.

As previously mentioned, the G2/M drug such as taxane derivative,preferably paclitaxel, will be administered in a similar regimen with aG1 and/or S phase drug such as β-lapachone or a derivative thereof,although the amounts will preferably be reduced from that normallygiven. It is preferred that for example the taxane be administered atthe same time as for example the β-lapachone or after the β-lapachonehas been given to the patient, typically about 24 hours after theβ-lapachone has been administered.

The other component of the combination therapy described is β-lapachoneor a derivative or analog thereof.

β-lapachone (3,4-dihydro-s,3-dimethyl-2H-naphthol[1,3-b]pyran-5,6-clone) is a simple plant product with a chemical structuredifferent from currently used anti-cancer drugs. It is obtained bysulfuric acid treatment of the naturally occurring lapachol, which isreadily isolated from Tabebuia avellanedae growing mainly in Brazil, oris easily synthesized from lomatiol, isolated from seeds of lomatiagrowing in Australia (Hooker, S., et al., J. Am. Chem. Soc.,58:1181-1190 (1936); Goncalves de Lima, O., et al., Rev. Inst. Antibiot.Univ. Recife., 4:3-17 (1962)).

β-lapachone has been shown to have a variety of pharmacological effects.β-lapachone is a topoisomerase I inhibitor but acts by a differentmechanism than camptothecin (Li, C. J., et al., J. Biol. Chem.,268:22463-22468 (1993). Numerous β-lapachone derivatives have beensynthesized and tested as anti-viral and anti-parasitic agent(Goncalves, A.M., et al., Mol. Biochem. Parasitology, 1:167-176 (1980);Schaffner-Sabba, K., et al., J. Med. Chem., 27:990-994 (1984); Li, C.,et al., Proc. Natl. Acad. Sci. USA, 90: 1842 (1993)). β-lapachone andits derivatives, e.g. 3-allyl-β-lapachone, show anti-trypanosomaleffects (Goncalves, A. M., et al., supra), the mechanism of which isunclear. β-lapachone has also been shown to be a DNA repair inhibitorwhich sensitizes cells to DNA damaging agents (Boorstein, R. J., et al.,Biochem. Biophys. Res. Commun., 118:828-834, (1984); Boothman, D. A., etal., J. Cancer Res., 49:605-612 (1989)). β-lapachone is well toleratedin dogs, rats, mice, and chickens. The maximum tolerated dose, whengiven p.o. daily for one month, is 200 mg/kg in rats, and 100 mg/kg indogs. Higher doses cause gastric ulceration and loss of erythrocytes,but not signs of bone marrow suppression (Ciba-Geigy, personalcommunication).

β-lapachone derivatives and analogs are known in the art and aredisclosed for example, in U.S. Pat. No. 5,828,700; WO97/08162; and U.S.Pat. No. 5,763,625. Preferred derivatives and analogs include compoundsof the following formulae I and II.

wherein R and R₁ are each independently selected from the groupconsisting of hydrogen, hydlroxy, thio (SH), halogen (e.g. fluoro,chloro and bromo), substituted and unsubstituted aryl, substituted andunsubstituted alkenyl, substituted and unsubstituted alkyl andsubstituted and unsubstituted alkoxy, and salts thereof, wherein thedotted double bond between the ring carbons to which R and R₁ are bondedrepresent an optional ring double bond. The alkyl groups preferably havefrom 1 to about 15 carbon atoms, more preferably from 1 to about 10carbon atoms, still more preferably from 1 to about 6 carbon atomis. Asused herein, the term alkyl unless otherwise modified refers to bothcyclic and noncyclic groups, although of course cyclic groups willcomprise at least three carbon ring members. Straight or branched chainnoncyclic alkyl groups are generally more preferred than cyclic groups.Straight chain alkyl groups are generally more preferred than branched.The alkenyl groups preferably have from 2 to 15 carbon atoms, morepreferably from 2 to about 10 carbon atoms, still more preferably from 2to about 6 carbon atoms. Especially preferred alkenyl groups have 3carbon atoms (i.e., 1-propenyl or 2-propenyl), with the allyl moietybeing particularly preferred. Phenyl and naphthyl are generallypreferred aryl groups. Alkoxy groups include those alkoxy groups havingone or more oxygen linkage and preferably have from 1 to 15 carbonatoms, more preferably from 1 to about 6 carbon atoms. Said substitutedR and R₁ groups may be substituted at one or more available positions byone or more suitable groups such as, for example, alkyl groups such asalkyl groups having from 1 to 10 carbon atoms or from 1 to 6 carbonatoms, alkenyl groups such as alkenyl groups having from 2 to 10 carbonatoms or 2 to 6 carbon atoms, aryl groups having from 6 to 10 carbonatoms, halogen such as fluoro, chloro and bromo, and N, O and S,including heteroalkyl, e.g., heteroalkyl having one or more of saidhetero atom linkages (and thus including alkoxy, aminoalkyl andthioalkyl) and from 1 to 10 carbon atoms or from 1 to 6 carbon atoms.

Compounds of formulae I and II can readily be made or obtained. (SeePardee, A., et al., Cancer Research, 49, 1-8 (1989); Schaffner-Sabba, K,et al., Journal of Medicinal Chemistry, 27, no. 8 990-994 (1984); S.Hooker, 58, 1181-1197 (1936).

Preferred compounds of formula I include β-lapachone,3-allyl-β-lapachone, 3-bromo-β-lapachone and 3-OH-β-slapachone.3-allyl-β-lapachone and 3-bromo-β-lapachone are more preferred.

Preferred compounds of formula II include 3-bromo-alpha-lapachone.

β-lapachone analogs of formula III, set forth below, can also be used inthe compositions and methods of the present invention.

where R is (CH₂)._(n)—R₁

where n is an integer from 0-10 and R₁ is hydrogen, an alkyl, an aryl, aheteroaromatic, a heterocyclic, an aliphatic, an alkoxy, a hydroxy, anamine, a thiol, an amide, or a halogen side group.

Preferred analogs of formula III include,3-ethoxycarbonylmethyl-β-lapachone, 3-(2′-Hydroxyethyl)-β-lapachone3-methyl-β-lapachone, 3-(2′-aminoethyl)-β-lapachone,3-methoxy-β-lapachone, 3-benzyloxy-β-lapachone,3-ethoxycarbonylmethoxy-β-lapachone and 3-allyloxy-β-lapachone.

Analogs of formula III can be produced by the methods disclosed in U.S.Pat. No. 5,763,625.

β-lapachone derivatives of formulae IV and V, set forth below, canfurther be used in the compositions and methods of the presentinvention.

wherein R¹-R⁶ are each, independently, selected from the groupconsisting of H, C₁-C₆ alkyl, C₁-C₆ alkenyl, C₁-C₆ alkoxy, C₁-C₆alkoxycarbonyl, —(CH₂)_(n)-aryl, (CH₂)_(n)-heteroaryl,—(CH₂)_(n)-heterocycle, and —(CH₂)_(n)-phenyl; or R¹ and R² combined area single substituent selected from the above group, and R³ and R⁴combined are a single substituent selected from the above group, inwhich case—is a double bond; and R⁷ is H, OH, C₁-C₆ alkyl, C₁-C₆alkenyl, C_(1-C) ₆ alkoxy, C₁-C₆ alkoxycarbonyl, —(CH₂)_(n)-amino,—(CH₂)_(n)-aryl, —(CH₂)._(n)-heteroaryl, —(CH₂)_(n)-heterocycle, or—(CH₂)._(n)-phenyl, wherein n is an integer from 0 to 10.

Preferred analogs of formulae IV and V include 3-(β-alanyl)-β-lapachoneand 3-malonyl-β-lapachone.

Analogs of formulae IV and V can be produced by the methods disclosed inU.S. Pat. No. 5,824,700.

Under the combination therapies described here, β-lapachone or aderivative or analog thereof is administered to a patient in at leastone dose in the range of 10 to 500,000 μg per kilogram body weight ofrecipient per day, more preferably in the range of 1000 to 50,000 μg perkilogram body weight per day, most preferably in the range of 5000 to25,000 μg per kilogram body weight per day. The desired dose is suitablyadministered once or several more sub-doses administered at appropriateintervals throughout the day, or other appropriate schedule. Thesesub-doses may be administered as unit dosage forms, for example,containing 1 to 20,000 μg, preferably 10 to 10,000 μg per unit dosageform.

As with the use of other chemotherapeutic drugs, the individual patientwill be monitored in a manner deemed appropriate by the treatingphysician. Typically, no additional drug treatments will occur until,for example, the patient's neutrophil count is at least 1500 cells/mm³.Dosages can also be reduced if severe neutropenia or severe peripheralneuropathy occurs, or if a grade 2 or higher level of mucositis isobserved, using the Common Toxicity Criteria of the National CancerInstitute.

The combination therapy agents described here may be administered singlyor in a cocktail containing both agents or one of the agents with othertherapeutic agents, including but not limited to, immunosuppressiveagents, potentiators and side-effect relieving agents. As aforesaid, thetherapeutic combination, if administered sequentially, is more effectivewhen the β-lapachone component is administered prior to the taxanederivative. The therapeutic agents will preferably be administeredintravenously or otherwise systemically by injection intramuscularly,subcutaneously, intrathecally or intraperitoneally.

The pharmaceutical compositions of this invention which are found incombination may be in the dosage form of solid, semi-solid, or liquidsuch as, e.g., suspensions, aerosols or the like. Preferably thecompositions are administered in unit dosage forms suitable for singleadministration of precise dosage amounts. The compositions may alsoinclude, depending on the formulation desired,pharmaceutically-acceptable, nontoxic carriers or diluents, which aredefined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, physiological saline, Ringer'ssolution, dextrose solution, and Hank's solution. In addition, thepharmaceutical composition or formulation may also include othercarriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenicstabilizers and the like. Effective amounts of such diluent or carrierwill be those amounts which are effective to obtain a pharmaceuticallyacceptable formulation in terms of solubility of components, orbiological activity, and the like.

For the purposes of the present invention, the G1 and/or S phasecompounds, derivatives or analogs and G2/M compounds, derivatives oranalogs described herein include their pharmacologically acceptablesalts, preferably sodium; analogs containing halogen substitutions,preferably chlorine or fluorine; analogs containing ammonium orsubstituted ammonium salts, preferably secondary or tertiary ammoniumsalts; analogs containing alkyl, alkenyl, aryl or their alkyl, alkenyl,aryl, halo, alkoxy, alkenyloxy substituted derivatives, preferablymethyl, methoxy, ethoxy, or phenylacetate; and natural analogs such asnaphthyl acetate. Further, the G and/or S phase compounds, derivativesor analogs and G2/M compounds, derivatives or analogs described hereinmay be conjugated to a water soluble polymers or may be derivatized withwater soluble chelating agents or radionuclides. Examples of watersoluble polymers are, but not limited to: polyglutamic acid polymer,copolymers with polycaprolactone, polyglycolic acid, polyactic acid,polyacrylic acid, poly (2-hydroxyethyl 1-glutamine), carboxymethyldextran, hyaluronic acid, human serum albumin, polyalginic acid or acombination thereof Examples of water soluble chelating agents are, butnot limited to: DTPA (diethylenetriaminepentaacetic acid), EDTA, DTTP,DOTA or their water soluble salts etc. Examples of radionuclides are,but not limited to: ¹¹¹In, ⁹⁰Y, ¹⁶⁶Ho, ⁶⁸Ga, ^(99m)Tc, etc.

In therapeutic applications, the dosages of the agents used inaccordance with the invention vary depending on the agent, the age,weight, and clinical condition of the recipient patient, and theexperience and judgment of the clinician or practitioner administeringthe therapy, among other factors affecting the selected dosage.Generally, the dose should be sufficient to result in slowing, andpreferably regressing, the growth of the tumors and also preferablycausing complete regression of the cancer. An effective amount of apharmaceutical agent is that which provides an objectively identifiableimprovement as noted by the clinician or other qualified observer.Regression of a tumor in a patient is typically measured with referenceto the diameter of a tumor. Decrease in the diameter of a tumorindicates regression. Regression is also indicated by failure of tumorsto reoccur after treatment has stopped.

This invention further includes pharmaceutical combinations comprising ataxane derivative and a dose of β-lapachone or a derivative or analogthereof as provided above and kits for the treatment of cancer patientscomprising a vial of the taxane derivative and a vial of β-lapachone ora derivative or analog thereof at the doses provided above. Preferably,the kit contains instructions describing their use in combination.

The documents mentioned herein are incorporated herein by reference.

It is understood that the foregoing detailed description and thefollowing examples are illustrative only and are not to be taken aslimitations upon the scope of the invention. Various changes andmodifications to the disclosed embodiments, which will be apparent tothose skilled in the art, may be made without departing from the spiritand scope of the present invention. Further, all patents, patentapplications and publications cited herein are incorporated herein byreference.

EXAMPLES in Vivo Testing

Experiment 1

Brief description of the tumor model (Cannistra model, Cannistra, etal., Cancer Res., 57:1228-1232 (1997)—Ovary cancer is a highly lethaldisease. Metastases occurs predominantly by widespread implantationthroughout the abdominal cavity. To test the efficacy of β-lapachonealone and in combination with TAXOL®, we used human ovary cancer ceUs(36M2) that were originally derived from a patient with malignantascites. Inoculation of these cells into female nude mice recapitulatesthe metastatic process as observed in patients. This is a highlymetastatic and malignant cancer cell model. In general, tumor nodules onperitoneum and malignant ascites develop 4 to 5 weeks after aninoculation of 10×10⁶ cells. Metastatic foci can be seen from one weekafter inoculation.

Animals—Athymic female nude (nulnu) were used throughout theexperiments.

Drugs—β-Lapachone was formulated into solution by using lipidol, amedium agent used clinically. Our success with this formulating agent(lipidol) solved the long-standing problem of insolubility ofβ-lapachone.

Alternatively, we also find that β-lapachone can be formulated incremphor plus ethanol [2 (cremphor):1 (ethanol)]. A solution of 20 mg/mlcan be prepared at room temperature.

TAXOL® (Ciba-Geigy) formulated solution (patient use standard) waspurchased from pharmacy and diluted with Lipidol (SumitomePharmaceuticals, Osaka) for the mouse experiment.

Both β-lapachone and TAXOL® solution can be administered eitherintraperitoneally or intravenously. Intraperitoneally routes for bothdrugs were used. Lipidol formulation of β-lapachone was used.

Design of Animal Trials:

Six Mice per Group.

Group 1, treated with control vehicle (as in group 4);

Group 2, treated with β-lapachone at 50 mg/kg;

Group 3, treated with TAXOL® at 0.1 mg/kg

Group 4, β-lapachone at 50 mg/kg first, followed by TAXOL® at 0.1 mg/kgthe next day. Repeat the cycle after two days.

All treatments were started a week after tumor inoculation. Mice weretreated for 10 cycles in total, and were sacrificed for tumor nodulecounting two weeks (on day 50) after discontinuation of drug treatment.

Upon sacrifice, antitumor activity in each group was evaluated bycounting the number of tumor nodules in the peritoneal cavity, measuringthe diameter of the tumors, measuring the volume of the ascites andqualitatively observing the color of the peritoneal wall as anindication of the degree of tumor-induced vascularization.

Toxicity was evaluated by qualitative observation of the generalappearance and behavior of the mice prior to sacrifice and by measuringtheir body weight at various intervals during the course of thetreatments.

The results of this experiment are set forth in FIGS. 1A and 1B. As canbe seen, the combination of β-lapachone and TAXOL® dramatically reducedthe number of tumor implants compared to the untreated control group andthe groups treated with each agent alone. No toxicity was observed.

Experiment 2

Tumor models and drug formulation were identical to Experiment 1. Again,6 mice were used for each group. The experimental design has thefollowing modifications: 1) TAXOL® dose were increased by 10 fold; 2)mice were observed for four weeks after the cessation of the drugtreatment.

Group 1, treated with control vehicle (as in group 4);

Group 2, treated with β-lapachone at 50 mg/kg;

Group 3, treated with TAXOL® at 1 mg/kg

Group 4, β-lapachone at 50 mg/kg first, followed by TAXOL® 1 mg/kg thenext day. Cycle repeated after two days.

All treatments were started one week after tumor inoculation. Mice weretreated for 10 cycles in total, and were sacrificed for tumor countingon day 62.

The results of this experiment are set forth in FIGS. 2A and 2B. As withexperiment 1, the combination of β-lapachone and TAXOL® dramaticallyreduced the number of tumor implants compared to the untreated controlgroup and the groups treated with each agent alone. No toxicity wasobserved.

FIGS. 3-6 visually represent the results seen in each group. In FIGS.3-5 the peritoneal lining is red (color not shown) which is indicativeof tumor stimulated blood vessel growth or angiogenesis.

The untreated mammals contain hundreds of tumor modules. Some of thenodules have fused together and formed large tumor masses (FIG. 3).

Treatment with either β-lapachone (FIG. 4) or TAXOL® (FIG. 5) resultedin a lessening of the tumors. However, the flesh in both areas was stillred, signing that substantial angiogenesis associated with the tumorswas still going on. Several tumor nodules were still present in bothgroups treated with single therapeutic segments.

In the group receiving combination therapy, the color of the flesh is nolonger bright red, but similar to that seen in healthy mice (FIG. 6).This indicates that the tumor induced angiogenesis seen in the singletreatment groups is substantially inhibited or not occurring. One or twosmall nodules were visually observed, but are not visible in thephotograph.

Experiment 3

Potent inhibition of prostate tumor growth in vivo by β-lapachone andtaxol. Male SCID (ICR) mice were inoculated with androgen-independenthuman prostate cancer cells (DU145; 8×10⁶ s.c.). Administration of drugswas initiated when tumor nodules reached ˜0.5 cm in diameter. Four miceper group were used in this experiment. The control group (FIG. 7A) wastreated with vehicle alone. The β-lapachone alone group was treated with50 mgikg i.p., and the taxol alone group was treated with 1 mg/kg i.p.,followed 24 h later by i.p. injection of vehicle. In the combinationgroup (FIG. 7B), mice were treated with β-lapachone alone, followed 24 hlater by taxol at 1 mg/kg. There was a 1-day break between each cycle.Mice were treated for a total of six cycles. Pictures were taken 3 weeksafter six cycles of treatment.

In preliminary experiment with four mice per group, andogen-independentDU145 prostate cancer cells were xenografted into immunocompromised mice(FIG. 7A). Again, to increase stringency and unlike most antitumorexperiments, treatment was delayed until the tumors reached ˜0.5 cm indiameter. Either β-lapachone or taxol alone showed moderate inhibitionsof tumor growth (data not shown). β-Lapachone plus taxol showed dramaticantitumor activity (FIG. 7B). Furthermore, tumors in the treated micedid not grow back as of the follow-up 6 weeks after treatment.

Experiment 4

Cell Cultures. All cell lines used in this study were obtained from theAmerican Type Culture Collection unless specified otherwise. Cells weremaintained at 37° C. in 5% CO₂ in complete humidity. Human breast cancercell lines MCF-7, 21 MT, 21 PT, and 21 NT (kindly provided by R. Sager,Dana-Farber Cancer Institute) were cultured in MEM-α (LiffeTechnologies, Grand Island, N.Y.), supplemented with 10% (vol/vol) FCS,2 mM L-glutamine, and 1 mg/ml insulin. Human ovary carcinoma cell linesAD2780s and AD2780DDP, a generous gift from K. J. Scanlon (City of HopeMedical Center, Duarte, Calif.); human colon adenocarcinoma cell linesSW1116, HT-29, and DLD; human lung carcinoma cell line G480; humanmelanoma cell line Skmel-28, kindly provided by G. Dranoff, (Dana-FarberCancer Institute); and human prostate tumor cell lines PC-3, DU145, andLNCaP were cultured in DMEM (Life Technologies) supplemented with 10%(vol/vol) FCS and 2 mM L-glutamine. Human pancreatic cancer cell lineASPC-1 was cultured in RPMI medium 1640 supplemented with 20% (vol/vol)FCS.

Colony Formation Assay. Exponentially growing cells were seeded at 1,000cells per well in six-well plates and allowed to attach for 48 h. Drugswere added directly to the dishes in less than 5 μl of concentratedsolution (corresponding to a final DMSO concentration of less than0.1%). Control plates received the same volume of DMSO alone. After 1-4h, cells were rinsed, and fresh medium was added. Cultures were observeddaily for 10-20 days and then were fixed and stained with modifiedWright-Giemsa stain (Sigma). Colonies of greater than 30 cells werescored as survivors.

Cell Death Assay. Cell death was determined by the KWT (Thiazolyl blue)assay or by trypan blue exclusion as indicated. Briefly, cells wereplated in a 96-well plate at 10,000 cells per well, cultured for 48 h incomplete growth medium, then treated with β-lapachone for 4 h, andcultured with drug-free medium for 24 h. MIT solution was added to theculture medium, and after 2 h. optical density was read with an ELISAreader. For the trypan blue exclusion assay, cells were cultured andtreated in the same way. They were harvested, and trypan blue dyesolution was added to the cell suspension. Total cell counts and viablecell numbers were determined with a hemocytometer.

Apoptosis Assays. Apoptosis was determined by three independent assays.One determined the sub-G₁ fraction of propidium iodide-stained nuclei asdescribed [Li, Y.-Z, et al. Mol. Med. 5:232-239 (1999); Li, C. J.,etal., Science 268:429-431 (1995); Li, CJ., et al., Cancer Res.55:3712-3715 (1995). The annexin assay measured the membrane changesdetermined by the externalization of phosphatidylerine (Fadok, V. A., etal., J. Immunol. 148:2207-2216 (1992). The third assay, analysis of DNAladdering, was carried out as described by Li, Y.-Z, supra.

Synergistic induction of cell death by β-lapachone and taxol. Colonyformation was carried out as described above. In a typical experiment,control DU145 cells in well 1 were treated with solvent on days 1 and 2.Cells in well 2 were treated with β-lapachone at 4 μM on day 1 for 4 h,incubated in drug-free medium for 20H, and then treated with solventcontrol on day 2. Taxol-along well 3 was treated with solvent controlfor 4 h on day 1 and with taxol at 0.02 μM for 4 h on day 2. Cells inwell 4 were treated with β-lapachone on day 1 and with taxol on day 2.In well 5, cells were treated with taxol on day 1 and with β-lapachoneon day 2. In well 6, cells were treated with β-lapachone and taxol onday 2.

Synergism of the Drug Combination. Colony formation of DU145 cells inthe control dish (well 1) was abolished when both taxol and β-lapachonewere applied. It was decreased only partly when taxol along (well 2) orβ-lapachone alone (well 3) were applied. To determine whether the orderof drug addition affects this observed powerful synergism of cellkilling, we varied the treatment schedule. A similar synergism wasobserved when cells were treated with taxol and β-lapachonesimultaneously (well 6) or with β-lapachone followed by taxol (well 4).Synergism was not observed if taxol was added before β-lapachonetreatment (well 5). This schedule dependency was observed in all thecell lines. These results suggest that the order of artificialcheckpoint imposition is important for the synergism mechanism.

Ablation of in Vitro Colonies in a Wide Spectrum of Human CarcinomaCells by the Combination of β-Lapachone and Taxol. Human carcinoma celllines of different histotypes were used to determine cell survival inthe colony formation assay (Table 2). The combination of β-lapachone andtaxol dramatically reduced cell survival in a variety of human cancercells, including ovarian, breast, prostate, melanoma, lung, andpancreatic cancer cell lines. β-Lapachone or taxol alone at theconcentrations used were much less effective in decreasing cancer cellcolony formation. This decreased cell survival was achieved by inductionof cell death as determined by the MTT (Thiazolyl blue) and trypan blueassays. Cell death was by apoptosis as determined by DNA ladderingformation and by annexin staining (data not shown). Taxol was at least10-fold more potent in the presence of β-lapachone, as measured at IC₅₀(data not shown).

TABLE 2 Inhibition of cancer cell survival by β-lapachone and taxolColonies, percentage of control β-Lapachone Cell line Tissue originsβ-Lapachone Taxol + taxol A2780DDP Ovary 77 (1.1) 39 (0.8) 0 MCF-7Breast 46 (1.4) 45 (0.3) 0 21MT Breast 56 (5.0) 63 (7.0) 0 Skmel-28Melanoma 56 (1.4) 44 (5.1) 0 HT-29 Colon 42 (1.4) 64 (2.5) 0 ASPC-1Pancreas 45 (1.9) 71 (0.8) 0 G480 Lung 32 (0.3) 39 (2.6) 2 (0.1) DU145Prostate 50 (2.2) 30 (0.9) 0

Cells were treated for 4 h with β-lapachone and/or taxol at thefollowing concentrations: A2780DDP, β-lapachone at 2 μM and/or taxol at0.2 μM; MCF-7 and 21-MT, β-lapachone at 4 μM and/or taxol at 0.1 μM;HT-29, β-lapachone at 4 μM; G480, β-lapachone at 4 μM and/or taxol at0.2 μM; DU145, β-lapachone at 4 μM and/or taxol at 0.2 μM. The number ofcolonies in control well was taken as 100% survival. Treated wells arepresented as percentage of control. Data are given as average (+SEM)from three independent experiments.

Although the foregoing invention has been described in some detail byway of illustration and example for the purposes of clarity ofunderstanding, one skilled in the art will easily ascertain that certainchanges and modifications may be practiced without departing from thespirit and scope of the appended claims.

What is claimed is:
 1. A method of treating a mammal having a solidtumor (or tumors) formed as a result of a cancer selected from the groupconsisting of melanoma, colon cancer, prostate cancer, lung cancer,pancreatic cancer, ovarian cancer and breast cancer, the methodcomprising: a) administering to the mammal an effective amount of afirst compound comprising β-lapachone or derivatives thereof as theactive ingredient; and b) administering to the mammal an effectiveamount of a G2/M phase drug.
 2. The method of claim 1, wherein the G2/Mphase drug is selected from the group consisting of microtubuletargeting and topoisomerase poison drugs.
 3. The method of claim 2,wherein the microtubule targeting drug is selected from the groupconsisting of taxol, docetaxel, vincristin, vinblastin, nocodazole,epothilones and navelbine.
 4. The method of claim 2, wherein thetopoisomerase poison drug is selected from the group consisting ofteniposide, etoposide, adriamycin, camptothecin, daunorubicin,dactinomycin, mitoxantrone, amsacrine, epirubicin and idarubicin.
 5. Themethod of claim 1 or 2, wherein the G2/M phase drug is taxol or a taxanederivative.
 6. The method of claim 1 or 2, wherein the G2/M phase drugis taxol.
 7. The method of claim 1 or 2, wherein the G2/M phase drug isadministered after the first compound.
 8. The method of claim 5, whereinthe taxane derivative is paclitaxel.
 9. The method of claim 5, whereinthe taxane derivative is paclitaxel and it is administeredintravenously.
 10. The method of claim 5, wherein the taxane derivativeis paclitaxel and it is administered intravenously after administrationof the β-lapachone first.
 11. A kit for the treatment of a mammaliantumor comprising separate vials containing β-lapachone or a derivativeor analog thereof and a taxane derivative, with instructions foradministering β-lapachone first.
 12. The kit of claim 11, wherein thetaxane derivative is paclitaxel.
 13. A pharmaceutical compositioncomprising β-lapachone or a derivative or analog thereof and a taxanederivative and a pharmaceutically acceptable carrier.
 14. Apharmaceutical composition of claim 13, where the taxane derivative ispaclitaxel.